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Cited by 134Hematopoietic Stem Cell TransplantationBlood disorders and treatmentsMesenchymal stem cell research

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Intrinsic and Extrinsic Control of Hemopoietic Stem Cell Numbers: Mapping of a Stem Cell Gene

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Haan, Gerald de, Zant, Gary Van·1997·The Journal of Experimental Medicine
DOI 10.1084/jem.186.4.529PMC PMC2199035
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Abstract

We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.

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Figure 1 The hemopoietic stem cell hierarchy as defined by the in vitro CAFC assay and its relationship with other stem/progenitor cell assays. Stem cells of a particular developmental stage form a cobblestone-like colony beneath a stromal cell layer after a defined period of time. Colonies that appear early (7 d) after initiation of the culture are generated by relatively mature progenitor cells, such as CFU-spleen (CFU-S) days 8 and 9, CFU–granulocyte/erythroid/megakaryocyte/macrophage (CFU-GEMM), CFU–granulocyte/macrophage (CFU-GM), or burst forming units–erythroid (BFU-E). Colonies which appear after ∼14 d in culture are descending from CFU-S days 12–14 or high proliferative potential–colony forming cells (HPP-CFC). In contrast, colonies which appear only after 5 wk in culture are correlated with very primitive, long-term repopulating ability (LTRA) or long-term culture initiating (LTC-IC) stem cells (18, 19). The frequency of stem cell subsets detected in vitro decreases exponentially with culturing time, reflecting the hierarchical structure of the stem cell compartment.
Figure 1 The hemopoietic stem cell hierarchy as defined by the in vitro CAFC assay and its relationship with other stem/progenitor cell assays. Stem cells of a particular developmental stage form a cobblestone-like colony beneath a stromal cell layer after a defined period of time. Colonies that appear early (7 d) after initiation of the culture are generated by relatively mature progenitor cells, such as CFU-spleen (CFU-S) days 8 and 9, CFU–granulocyte/erythroid/megakaryocyte/macrophage (CFU-GEMM), CFU–granulocyte/macrophage (CFU-GM), or burst forming units–erythroid (BFU-E). Colonies which appear after ∼14 d in culture are descending from CFU-S days 12–14 or high proliferative potential–colony forming cells (HPP-CFC). In contrast, colonies which appear only after 5 wk in culture are correlated with very primitive, long-term repopulating ability (LTRA) or long-term culture initiating (LTC-IC) stem cells (18, 19). The frequency of stem cell subsets detected in vitro decreases exponentially with culturing time, reflecting the hierarchical structure of the stem cell compartment.CC BY-NC-SA · PubMed Central
Figure 2 Individual mouse lifespan is negative correlated with the number of cycling CAFC day 7 per femur. The fraction of cells in S-phase was determined by using an in vitro HU suicide technique. The linear regression equation was: CAFC day 7 cycling = 13159 − 18.47 × lifespan, r 2 = 0.66, P <0.0145.
Figure 2 Individual mouse lifespan is negative correlated with the number of cycling CAFC day 7 per femur. The fraction of cells in S-phase was determined by using an in vitro HU suicide technique. The linear regression equation was: CAFC day 7 cycling = 13159 − 18.47 × lifespan, r 2 = 0.66, P <0.0145.CC BY-NC-SA · PubMed Central
Figure 3 Different strains of mice have largely varying primitive stem cell numbers. CAFC subsets were quantified in eight inbred strains of mice for five consecutive weeks. Each point represents data obtained from 6 to 15 mice in a minimum of two independent experiments. CAFC frequency is shown on a logarithmic scale.
Figure 3 Different strains of mice have largely varying primitive stem cell numbers. CAFC subsets were quantified in eight inbred strains of mice for five consecutive weeks. Each point represents data obtained from 6 to 15 mice in a minimum of two independent experiments. CAFC frequency is shown on a logarithmic scale.CC BY-NC-SA · PubMed Central
Figure 4 Inbred strains of mice can be divided into three groups with respect to the organization of their stem cell compartment: group 1: CBA, C3H, and BALB/c (high cycling, few stem cells, short lifespan); group 2: C57BL/6 and 192/Sv (low cycling, intermediate stem cells, long lifespan); and group 3: DBA and AKR (high cycling, many stem cells, short lifespan). Left y-axis shows mean lifespan for each strain (•), right y-axis shows mean number of cycling CAFC day 7 per femur (★).
Figure 4 Inbred strains of mice can be divided into three groups with respect to the organization of their stem cell compartment: group 1: CBA, C3H, and BALB/c (high cycling, few stem cells, short lifespan); group 2: C57BL/6 and 192/Sv (low cycling, intermediate stem cells, long lifespan); and group 3: DBA and AKR (high cycling, many stem cells, short lifespan). Left y-axis shows mean lifespan for each strain (•), right y-axis shows mean number of cycling CAFC day 7 per femur (★).CC BY-NC-SA · PubMed Central
Figure 5 Strain-specific responses to in vivo administration of FL. The number of cycling CAFC day 7 (a) and CAFC day 35 (b) was determined using an in vitro HU suicide technique. Dark bars represent the number of CAFC in S-phase in untreated control animals, while lighter bars show the cycling activity in FL-treated mice. Note that no detectable CAFC day 35 cycling could be demonstrated in any strain before FL administration, except AKR. Groups indicated refer to categories as shown in Fig. 4.
Figure 5 Strain-specific responses to in vivo administration of FL. The number of cycling CAFC day 7 (a) and CAFC day 35 (b) was determined using an in vitro HU suicide technique. Dark bars represent the number of CAFC in S-phase in untreated control animals, while lighter bars show the cycling activity in FL-treated mice. Note that no detectable CAFC day 35 cycling could be demonstrated in any strain before FL administration, except AKR. Groups indicated refer to categories as shown in Fig. 4.CC BY-NC-SA · PubMed Central
Figure 6 Analysis of stem cell frequency in RI strains. (a) Strain distribution pattern of the CAFC day 35 frequency in all BXD strains. Parental values are indicated with the leftmost (C57BL/6) and rightmost (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval mapping on chromosome 18. The threshold LRS values for suggestive and significant linkage were 9.9 and 14.7, respectively, and are indicated. A sharp peak was observed around D18NcvS22, located ∼19 cM from the centromere. The corresponding map position of orthologous genes in human is shown at the top of the figure. The region around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.
Figure 6 Analysis of stem cell frequency in RI strains. (a) Strain distribution pattern of the CAFC day 35 frequency in all BXD strains. Parental values are indicated with the leftmost (C57BL/6) and rightmost (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval mapping on chromosome 18. The threshold LRS values for suggestive and significant linkage were 9.9 and 14.7, respectively, and are indicated. A sharp peak was observed around D18NcvS22, located ∼19 cM from the centromere. The corresponding map position of orthologous genes in human is shown at the top of the figure. The region around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.CC BY-NC-SA · PubMed Central
Figure 7 Genes assigned to human chromosome 5q21-35, and their map position in mouse. The order of genes was based on various sources (26, 30, 32). The regions which have been implicated in human 5q associated malignancies are indicated, with references. The region which contains the QTL mapped in this study is shown in the last column.
Figure 7 Genes assigned to human chromosome 5q21-35, and their map position in mouse. The order of genes was based on various sources (26, 30, 32). The regions which have been implicated in human 5q associated malignancies are indicated, with references. The region which contains the QTL mapped in this study is shown in the last column.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Haan, G. D., & Zant, G. V.
Affiliations
  • University of Kentucky HealthCare (US)
  • From the Blood and Marrow Transplant Program, Division of Hematology/Oncology, University of Kentucky Medical Center, Lexington, Kentucky 40536-0093
ORCID
Haan, Gerald de
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1997-08-18
Volume
186
Issue
4
Pages
529-536
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.186.4.529
PMC
PMC2199035
OpenAlex
W2115154817
Cited by
134
References
33
Citations by year
2020: 2 · 2018: 4 · 2017: 1 · 2016: 2 · 2015: 6 · 2013: 3 · 2012: 3
FWCI
5.39
Type
journal article
Language
en
Open access
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • UKnowledge (University of Kentucky) (submittedVersion, cc-by-nc-sa)
Funders
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Topics
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
  • Health Sciences › Medicine › Hematology › Blood disorders and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
Keywords
hematopoietic stem cells, stem cell frequency, Flt3 ligand, stem cell quiescence, cell cycle kinetics, cobblestone area-forming cells, BXD recombinant inbred mice, linkage mapping, chromosome 18, EGR1, 5q- syndrome, mouse strain differences, mouse lifespan
MeSH terms
Animals, Cell Count, Cell Division, Chromosome Mapping, Female, Hematopoietic Stem Cells, Humans, Mice, Inbred Strains, Species Specificity, Mice

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